Cancer has become the leading cause of morbidity and death worldwide, with annual deaths expected to reach 10.3 million by 2020. Recent epidemiological studies have shown that a dramatic rise in obesity and the associated type 2 diabetes are major risk factors for cancer. These observations have stimulated the critical need to identify the molecular signaling pathways that drive tumorigenesis in response to the obese setting, such that novel therapies can be developed. Although increased fats and carbohydrates are commonly cited as the major factors in diet-induced dysmetabolic states, increased protein consumption has also emerged as a potential contributor, acting through elevated levels of circulating branched-chain amino acids (BCAAs). BCAA transporters are upregulated in many cancer types and their increased expression correlates with the stage of malignancy, suggesting that increased BCAA availability and enhanced BCAA influx may be sufficient to drive tumorigenesis. The mamalian target of rapamycin complex1 (mTORC1) acts as a regulatory node by integrating input from nutrients and mitogens to positively control cell growth and proliferation. Critically, the mechanism by which BCAAs promote malignancy appears to be mechanistically dependent on BCAA-induced mTORC1 signaling. Class III phosphatidylinositol-3OH kinase (PI3K), a heterodimer consisting of the catalytic subunit, hVps34, and a regulatory subunit, hVps15, mediates the response to nutrient input to mTORC1. Class III PI3K is also required for Src- dependent tumorigenesis. The key role of class III PI3K in the mTORC1-dependent nutrient response and tumorigenesis suggests that targeting class III PI3K may prove to be a novel and efficacious anti- cancer therapy. The long-term goal of this proposal is to delineate the mechanisms through which the class III PI3K mediates nutrient-induced mTORC1 signaling and to examine the role of the class III PI3K in promoting tumorigenesis and tumor progression. The central hypothesis is that the tumorigenic role of class III PI3K is manifested through the mTORC1 signaling pathway and that class III PI3K is a candidate for therapeutic intervention. To address this central hypothesis, two specific aims will be pursued. Aim 1 is to test the hypothesis that the class III PI3K regulatory subunit, hVps15, positively regulates mTOR protein stability and cellular localization. Aim 2 is to test the hypothesis that hVps15 is necessary for tumorigenesis. These specific aims will be achieved through genetic manipulation of hVps15 in cell culture, assessment of mTORC1 signaling, and determination of mTOR protein half-life. In situ and in vivo models of tumorigenesis, in conjunction with RNAi-based disruption of hVps15 and pharmacological inhibition of mTOR, will be used to examine the role of the class III PI3K in tumorigenesis. PUBLIC HEALTH RELEVANCE: Deaths caused by cancer are continuing to rise worldwide, and are, in large part, thought to be accompanying the escalating obesity epidemic. Excess nutrients are strong drivers of cancer. Thus, this proposal aims to understand how nutrient-regulated signals promote cancer, and to obtain a deeper understanding of the nutrient-regulated signaling pathways themselves as a first step in developing more effective cancer therapies.